The DNA damage checkpoints are cellular surveillance mechanisms that allow the cell to monitor the integrity of the genome and to signal the presence of DNA damage to the cell. These pathways play a critical role in maintaining genomic stability, and loss of checkpoint function has been implicated in the development of cancer. Genomic instability and defects in some checkpoint genes have also been linked to several syndromes that lead to premature aging, including Werner's syndrome. The long-term objective of this research is to understand the mechanism by which cells signal the presence of different forms of DNA damage to the cell cycle machinery and DNA repair proteins. The focus of this application is ATR (ATM and Rad3-related), a mammalian gene that is required for the DNA damage and replication checkpoints. The primary goal is to identify domains in ATR and other proteins that are required for ATR to sense and transduce the presence of DNA damage to the cell. The 9-1-1 complex is a heterotrimeric protein complex that may be required for ATR to signal to its downstream effectors. The specific aims of this research are (1) to determine if the 9-1-1 complex is an upstream regulator and/or downstream effectors. The specific aims of this research are (1) to determine if the 9-1-1 complex is an upstream regulator and/or downstream effector of ATR function (2) to identify and characterize functional domains within ATR, and (3) to purify, clone and characterize the protein that mediates the interaction of ATR with DNA. Extracts from the eggs of Xenopus laevis will be used to study the relationship between the 9-1-1 complex and ATR. Using a series of assays that we developed, the function of a specific ATR fragment that exhibits a unique nuclear localization and a dominant negative phenotype will be probed in mammalian cells. Finally, using a new assay for ATR's DNA- binding activity and a new affinity-based purification strategy, the protein that mediates the interaction of ATR with DNA will be purified, cloned and characterized. The studies will provide a better understanding of the mechanisms by which ATR signals he presence of different forms of DNA damage to the cell. Furthermore, they should provide important insights into human disease such as cancer and the complex process of human aging.